Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 2/29/2016
Publication Date: 6/1/2016
Citation: Myer, P.R., Kim, M.S., Freetly, H.C., Smith, T.P. 2016. Evaluation of 16S Rrna amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers [abstract]. American Society for Microbiology. Poster No. SUNDAY-584.
Technical Abstract: Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. The objective of this study was to compare and analyze short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions,V1-V8, using highly diverse microbial communities from the rumen contents of steers (n=32), in order to examine the impact of technology selection on phyogenetic profiles. Short paired end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long "circular consensus" reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms predicted similar microbial operational taxonomic units (OTUs) as well as species richness, Good's coverage, and Shannon diversity. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater for the near full-length read (P < 0.05). UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the general consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. Partially funded by National Institute of Food and Agriculture Grant 2011-68004-30214 National Program for Genetic Improvement of Feed Efficiency in Beef Cattle.